human crispra pooled library set a  (Addgene inc)

Journal: Cancer Discovery

Article Title: Paradoxical Activation of Oncogenic Signaling as a Cancer Treatment Strategy

doi: 10.1158/2159-8290.CD-23-0216

Figure Lengend Snippet: LB-100 activates oncogenic signaling, engages stress response pathways, and restrains the proliferation of colorectal cancer cells. A , Gene set enrichment analyses on time-course transcriptome data from HT-29 and SW-480 cells show selected “Hallmarks” and “KEGG” molecular signatures modulated by LB-100 (4 μmol/L). Darker bars indicate time points for which the respective gene set was significantly enriched (P <0.05). B , Time-course western blots show selected oncogenic signaling and stress response pathways modulated by LB-100 (4 μmol/L) in HT-29 and SW-480 cells. α-Tubulin and Vinculin were used as loading controls. C , IncuCyte-based proliferation assays with the colorectal cancer models in the absence or presence of LB-100 at 1, 2, or 4 μmol/L for the indicated times. D , String network combining all hits identified by the two independent genome-wide CRISPR screens (Supplementary Fig. S2) as modulators of LB-100 toxicity. Only high-confidence interactions are shown and disconnected nodes are omitted. Green nodes: CRISPRa screen; orange nodes: CRISPR-KO screen; yellow node: identified on both screens. E , GO analyses using the full list of hits from both CRISPR screens (Supplementary Fig. S2) as input. The top 5 enriched Biological Processes and Molecular Functions terms are shown. Darker bars highlight WNT/β-catenin-and MAPK-related terms.

Article Snippet: HT29 dCas9-VP64 clone E cells were transduced with lentivirus of Calabrese pooled human CRISPRa library sets A and B (Addgene, 92379 and 92380) separately, in the presence of 8 μg/mL polybrene (Santa Cruz, sc-134220A), and at an MOI of approximately 0.3.

Techniques: Western Blot, Genome Wide, CRISPR

Journal: bioRxiv

Article Title: Paradoxical activation of oncogenic signaling as a cancer treatment strategy

doi: 10.1101/2023.02.06.527335

Figure Lengend Snippet: (A) Gene Set Enrichment Analyses on time-course transcriptome data from HT-29 and SW-480 cells show selected “Hallmarks” and “KEGG” molecular signatures modulated by LB-100 (4 µM). Darker bars indicate time points for which the respective gene set were significantly enriched (p-value <0.05). (B) Time-course western blots show selected oncogenic signaling and stress response pathways modulated by LB-100 (4 µM) in HT-29 and SW-480 cells. α-Tubulin and Vinculin were used as loading controls. (C) IncuCyte-based proliferation assays with the CRC models in the absence or presence of LB-100 at 1, 2, or 4 µM for the indicated times. (D) String network combining all hits identified by the two independent genome-wide CRISPR screens as modulators of LB-100 toxicity. Only high-confidence interactions are shown and disconnected nodes are omitted. Green nodes: CRISPRa screen; orange nodes: CRISPR-KO screen; yellow node: identified on both screens. (E) Gene Ontology (GO) analyses using the full list of hits from both CRISPR screens as input. The top 5 enriched Biological Processes and Molecular Functions terms are shown. Darker bars highlight WNT/β-Catenin- and MAPK-related terms.

Article Snippet: HT29 dCas9-VP64 clone E cells were transduced with lentivirus of Calabrese pooled human CRISPRa library set A and B (Addgene, 92379 and 92380) separately, in the presence of 8 μg/ml polybrene (Santa Cruz, sc-134220A), and at a multiplicity of infection of approximately 0.3.

Techniques: Western Blot, Genome Wide, CRISPR

Journal: bioRxiv

Article Title: Paradoxical activation of oncogenic signaling as a cancer treatment strategy

doi: 10.1101/2023.02.06.527335

Figure Lengend Snippet: (A) The bubble plot shows genes whose overexpression in the CRISPRa screen was selectively toxic for LB-100-treated (5 µM) cells comparing to untreated controls. 6 different gRNAs per gene were tested in 3 independent replicates. Cells on both conditions were grown for 12 population doublings before DNA harvesting and sequencing. Hits were called based on 0.25 false discovery rate (FDR) and at least 1 log2 fold-change difference between treated and untreated samples. FDR values < 10e-10 are truncated to 10e-10. Only the hits mentioned on the main text are named and colored, the full list of hits is presented on the . (B) The bubble plot shows genes whose knockout in the CRISPR-KO screen was enriched in LB-100 (6 µM) treated cells comparing to untreated (DMSO) controls. 4 different gRNAs per gene were tested in 3 independent replicates. Cells on both conditions were grown for at least 6 population doublings before DNA harvesting and sequencing. Hits were called based on 0.25 false discovery rate (FDR) and at least 1 log2 fold-change difference between treated and untreated samples. FDR values < 10e-10 are truncated to 10e-10. Only the hits mentioned on the main text are named and colored, the full list of hits is presented on the .

Article Snippet: HT29 dCas9-VP64 clone E cells were transduced with lentivirus of Calabrese pooled human CRISPRa library set A and B (Addgene, 92379 and 92380) separately, in the presence of 8 μg/ml polybrene (Santa Cruz, sc-134220A), and at a multiplicity of infection of approximately 0.3.

Techniques: Over Expression, Sequencing, Knock-Out, CRISPR

Journal: bioRxiv

Article Title: Paradoxical activation of oncogenic signaling as a cancer treatment strategy

doi: 10.1101/2023.02.06.527335

Figure Lengend Snippet:

Article Snippet: HT29 dCas9-VP64 clone E cells were transduced with lentivirus of Calabrese pooled human CRISPRa library set A and B (Addgene, 92379 and 92380) separately, in the presence of 8 μg/ml polybrene (Santa Cruz, sc-134220A), and at a multiplicity of infection of approximately 0.3.

Techniques: Over Expression, Comparison, Selection